title: Multiplex PCR-based detection of circulating tumor cells in lung cancer patients using CK19, PTHrP, and LUNX specific primers.
abstract: BACKGROUND
  The aim of this study was to develop a multiplex polymerase chain reaction (PCR)-based method for detection of circulating tumor cells in peripheral blood of lung cancer (LC) patients.
  
  
  METHODS
  Peripheral blood was collected from 71 healthy donors and 125 LC patients at different pathological stages. Samples were analyzed using multiplex PCR, and specific primers for CK19, PTHrP, and LUNX mRNA. The sensitivity of our method was set at 10 LC cells (A549 cells) in 3 mL of peripheral blood of healthy donors using spiking experiments.
  
  
  RESULTS
  The detection rates in LC patients for CK19, PTHrP, and LUNX were 45.6%, 64.8%, and 28%, and in healthy individuals were 7%, 7%, and 5.6%, respectively. Overall, our method produced 77.8% positive detections for at least 1 molecular marker. Twenty-eight (22.2%) were negative for expression of all markers, 39 (31.2%) were positive for expression of 1 marker, 42 (33.6%) were positive for expression of 2 markers, and 17 (13.6%) were positive for expression of all 3 markers. Detection of CK19 mRNA expression positively correlated with LC stage and distant metastases. PTHrP mRNA detection correlated positively with LC stage, presence of bone metastasis, and squamous cell carcinoma, and LUNX mRNA detection correlated with lymph node involvement. Combined detection of 2 or 3 markers was significantly correlated with metastatic disease, and negative detection of all 3 molecular markers was correlated with early stage nonmetastatic disease.
  
  
  CONCLUSIONS
  Multiple PCR-based detection of CK19, PTHrP, and LUNX mRNA expression provides useful information for disease stage and dissemination in LC patients.
authors.affiliation: Department of Experimental Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Katseli
authors.forename: Haralampos
authors.initials: H
authors.lastname: Maragos
authors.forename: Adrianos
authors.initials: A
authors.lastname: Nezos
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Syrigos
authors.forename: Michael
authors.initials: M
authors.lastname: Koutsilieris
mesh_terms: D000230:Adenocarcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics; D018287:Carcinoma, Large Cell / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D016022:Case-Control Studies; D005260:Female; D005500:Follow-Up Studies; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D053539:Keratin-19 / Q000235:genetics*; D008175:Lung Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics*; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055752:Small Cell Lung Carcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: C485599:BPIFA1 protein, human; D014408:Biomarkers, Tumor; D006023:Glycoproteins; D053539:Keratin-19; D044162:Parathyroid Hormone-Related Protein; D010750:Phosphoproteins; D012333:RNA, Messenger
keywords: Keratin 19; Lung-specific X protein; Multiplex PCR; Parathyroid hormone-related protein; Peripheral blood